Project Summary SAHA is an FDA-approved chemotherapeutic agent that has found success as a treatment for T- cell lymphoma. This histone deacetylase (HDAC) inhibitor is additionally part of over 240 past or ongoing clinical trials (clinicaltrials.gov) that seek to examine SAHA efficacy as a treatment option for many other cancer subtypes and pathologies. Despite widespread study at the clinical level, the therapeutic potential of SAHA and other HDAC inhibitors is hampered by our rudimentary understanding of the molecular mechanisms that mediate their actions. Our proposed studies will shed light upon such mechanisms and characterize the response of the SIN3 HDAC complexes to SAHA. Our objective is to characterize the functional dynamics of SIN3A and SIN3B complexes. We hypothesize that SIN3A and SIN3B complexes possess divergent functional attributes and are differentially responsive to HDAC inhibitors. Our objective will be addressed through two aims. In aim 1, we will characterize the biophysical properties of SIN3A and SIN3B complexes. To examine the SIN3A and SIN3B core complex architectural arrangements and responses to HDAC inhibitors, we will employ crosslinking mass spectrometry to identify sites of protein-protein interactions within the complex and monitor their responses to HDAC inhibitors. In aim 2, we will examine the unique roles held by SIN3A and SIN3B proteins within SH-SY5Y neuroblastoma and HEK293 cells and characterize their influences on biological pathways. In the process of addressing these central biological issues, we will make significant contributions to the field of quantitative proteomics by developing an assay system capable of quantifying the dynamics of protein-protein interactions via crosslinking mass spectrometry. As SAHA and other HDAC inhibitors are already components of clinical treatment plans, the proposed studies will produce information that will be immediately useful as we define the therapeutic potential of SAHA. Long- term, these findings will provide the groundwork for the development of HDAC inhibitors that target specific aspects of HDAC complexes and produce few off-target effects.